Detection of nucleic acids has grown in recent years as a means for early detection of genomic features, infectious agents and various organisms which are present in very small quantities in a human or animal test specimen. Detection procedures are normally based on the concept of complementarity whereby two DNA strands are bound together by hydrogen bonds and other forces between complementary nucleotides (which are known as nucleotide pairs).
A DNA molecule is normally quite stable, but the strands can be separated or denatured by certain conditions, such as heating. The denatured strands will reassociate only with another strand having a complementary sequence of nucleotides.
Much research has been carried out to find ways to detect only a few molecules of a DNA. Various procedures are known and have been used for almost a decade to amplify or greatly multiple the number of nucleic acids in a specimen for detection. Such amplification techniques include polymerase chain reaction (PCR), ligase chain reaction (LCR) and others which are less developed.
PCR is the most well known and involves the hybridization of primers to the strands of a target nucleic acid in the presence of a DNA polymerization agent and deoxyribonucleotide triphosphates under appropriate conditions. The result is the formation of primer extension products throughout several cycles and exponential multiplication of the number of original target strands. Further details about PCR can be obtained by consulting U.S. Pat. No. 4,683,195 (Mullis et al), U.S. Pat. No. 4,683,202 (Mullis) and U.S. Pat. No. 4,965,188 (Mullis et al).
Human and animal specimens contain many different nucleic acids, some of which are endogenous (or natural) to the person or animal, and others which are produced because of some abnormal condition, such as from the presence of an infectious agent or an oncogenic condition. Such nucleic acids are usually present in very low concentrations compared to endogenous nucleic acids. They are sometimes referred to as "low copy number" nucleic acids. By comparison, the endogenous nucleic acids are usually present in high concentrations and may be referred to as "high copy number" nucleic acids. One such example is human .beta.-globin DNA.
Frequently, in using PCR, two or more nucleic acids present in the specimen are amplified at the same time in the same reaction container. This is identified herein as "coamplification". This process requires that primers for each nucleic acid to be amplified must be simultaneously present in the container.
When both low and high copy target nucleic acids are amplified in such situations, amplification of the low copy target nucleic acid is often inhibited. This is due to the saturation of the amplifying enzyme (such as DNA polymerase) by the high copy target nucleic acid during the later cycles of amplification. False negative results for the presence of the low copy target nucleic acid would be likely, with possibly serious consequences.
Various solutions to the problem have been proposed for PCR, including adjusting the concentrations of the primers, utilizing primer sets with specific melting temperatures (Tm's), or combinations thereof. Adjusting the primer ratios has been referred in the art as "primer biasing" the PCR yield, and requires a decrease in the concentration of primers for the high copy target nucleic acid. Only modest control of the process is achieved with this approach.
Another approach to coamplification has been to adjust the temperature of annealing in PCR such that the primers for the high copy target nucleic acid anneal to a lesser extent than those for the low copy target nucleic acid. This approach also has a problem. The T.sub.m difference between primer pairs must be relatively large before good modulation of PCR can be exerted on the differential yields for the high and low copy nucleic acids. Exact T.sub.m 's cannot be calculated (although they can be estimated), and thus they must be measured. This requires a high degree of effort, and considerable tedium.
All of these approaches to modulate coamplification require that the high and low copy target nucleic acid sequences be known.
Alternatively, adding time to the priming or extension steps in PCR in later cycles can minimize the DNA polymerase saturation by the high copy target nucleic acid and increase amplification efficiency. However, this solution has limited utility in situations where many nucleic acids which are present in varying concentrations, are being amplified simultaneously.
It would be desirable to achieve rapid and efficient amplification of one or more low copy target nucleic acids when coamplified in the presence of one or more high copy target nucleic acids.